centromere identifier


EVOLUTIONARY HOMOLOGS

Evolutionary conservation of Cid in Drosophila species

Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, an excess of replacement changes have been found that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. It is proposed that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery (Malik, 2001).

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. Cid sequences have been compared from a phylogenetically broad group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. This analysis reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres (Malik, 2002).

In all 22 species studied, the Cid gene lacks introns and varies from 639 to 879 nucleotides in length. Most of the length variation in Cid can be attributed to the N-terminal tail, whereas the histone fold domain is nearly constant in length. A multiple alignment of the histone fold domain is presented along with the Drosophila H3 sequence for comparison. Most residues involved in mediating the histone-histone interactions of H3 are well conserved in Cid, consistent with their structural role. In contrast, the Loop 1 region of Cid, which is predicted to form an extensive DNA-interaction domain with Loop 2 of H4, is more variable. All CenH3s have a longer Loop 1 region, consistent with its predicted role in conferring some degree of DNA-binding specificity. The two replacements fixed between melanogaster and simulans in Loop 1 have been subject to adaptive evolution (Malik, 2001).

In the present analysis, comparison of pairs of closely related species indicate additional positions in Loop 1 that have undergone frequent episodes of amino acid replacements. For instance, two replacements separate yakuba from teissieri [GAT GAA GCA (amino acids DEA) versus GAT GGA GAA (DGE), respectively], whereas four replacements separate erecta from orena [TTG AGG GTC TCC GAG GGC (LRVSEG) versus TTG AAG ATC AGC GTG GCA (LKISVA), respectively] in their Loop 1 regions. The adaptive evolution that characterizes Loop 1 may be indicative of changing DNA specificity (Malik, 2001). In addition to regions subject to adaptive evolution, this analysis also highlights residues in Loop 1 that are well conserved and likely play a structural role in Cid function instead of conferring DNA specificity (Malik, 2002).

Based on the structure of the nucleosome, there are additional DNA-interaction sites of structural significance on the H3 molecule that are well conserved in Cid. From the multiple alignment, it was found that both the N-terminal helix alphaN and the carboxy-terminus of the protein are also evolving rapidly. alpha N is predicted to make specific contacts with the DNA gyres as the N-terminal tail of H3 exits the nucleosome and like Loop 1, this region may also confer some discrimination in DNA binding (Malik, 2002).

Based on a multiple alignment of the nucleotide sequences of the histone fold domain, the phylogeny of the Cid gene in the melanogaster group of species can be reconstructed. As expected for an essential single-copy gene, the phylogeny of Cid is congruent with previous phylogenies of this group of species. The divergence between the melanogaster species group and the ananassae species groups is estimated as at least 12.7 million years (based on paraphyly of ananassae, montium, and melanogaster species groups), whereas that between the obscura and melanogaster groups is estimated as 24 million years. Despite a total alignment length of only 300 nucleotides, a high resolution is observed in the phylogeny. Because eukaryotes are presumed to have one characteristic CenH3, it might serve as a universal phylogenetic marker in groups that have been traditionally hard to resolve (Malik, 2002).

In contrast to the histone fold domain, only a few segments of the N-terminal tail are conserved. Three stretches of conserved protein sequences are identified and been termed Blocks 1-3 respectively. Block 1 is 44 aa long and occurs close to the N-terminal end of the protein. Block 1 is unique in that other significant hits in the non-redundant database could not be found when using a MAST search. Block 2 is 20 aa long and occurs in the middle of the N-terminal tail. It primarily consists of a stretch of acidic residues flanked by a pair of well conserved serines. No homologous sequences to Block 2 could be found in the database other than those in very acidic proteins. Block 3 is 11 aa long and immediately abuts the histone fold domain. It is characterized by a basic region as well as invariant proline and arginine residues. The position of the third block is significant because the N-terminal tail of histone H3 is predicted to exit the nucleosome through a minor groove channel precisely at this junction. Block 3 resembles a putative nuclear localization signal encoded by four consecutive basic residues. There is precedent for some histones relying on an NLS-dependent pathway of nuclear import, but this has yet to be demonstrated for any CenH3 (Malik, 2002).

Most of the length polymorphism in different Cid genes can be directly traced to oligopeptide repeat expansions in the N-terminal tail. These oligopeptides vary both in repeat lengths as well as the number of iterations. Thus, Drosophila lucipennis has a pentameric glutamine (Q) expansion, whereas D. teissieri and D. orena have three repeats of (QN) and (NPKS), respectively. The diverse location of these expansions and their absence in closely related species attest to their independent evolutionary origins. In the lineages leading to the takahashii, suzukii and ananassae species groups, the expansions occur in the same location and are each 9 aa residues long. In the takahashii and suzukii sister groups, copy number varies from 1 to 3, whereas in the ananassae group, it varies from 3 to 6 copies (Malik, 2002).

To investigate the evolutionary significance of the oligopeptide repeat expansions in Cid's N-terminal tail, all instances of the repeat expansion in the takahashii/suzukii and ananassae species groups were aligned. From the multiple synonymous and replacement substitutions, it is evident that the repeats are ancient and have not been subject to concerted evolution (which would lead to low nucleotide diversity). Despite their age, the amino acid residues encoded at many of the nine positions are subject to purifying selection. Indeed, in comparisons of these repeats within strains of bipectinata, six synonymous and no replacement polymorphisms are found. The number of iterations of the repeat is not fixed in a species. For example, three strains of D. bipectinata have five copies of each repeat, whereas two strains have six. The Logo of the nine-residue stretch clearly highlights conserved and rapidly evolving residues. The two consensus oligopeptides from takahashii and ananassae groups are similar to each other in the last four positions (Malik, 2002).

The first five positions of the ananassae consensus are similar to previously defined four-residue-long SPKK motifs, known to mediate histone interactions with linker DNA in the minor groove. SPKK motifs have been found in the N-terminal tails of sea urchin sperm histones H1 and H2B, as well as the C-terminal tails of a class of angiosperm histone H2As. SPKK motifs are also found embedded in larger repeats within the carboxy-terminal tails of linker histone H1s (Malik, 2002).

The SPKK class of minor groove binding motifs includes substantial variation at the primary sequence level. The structure of SPKK bound to DNA has not been solved, but NMR and structural modeling suggest that SPKK forms a turn stabilized by one or two hydrogen bonds. The structure is called an Asx turn if the hydrogen bond is between the OH or CO side chain of the ith amino acid (S, T, D, or N) and the main chain NH of the i+2 amino acid (A or a hydrophilic amino acid; e.g., K,R,E,T). In a beta turn there is a hydrogen bond between the main chain CO of the ith amino acid and the main chain NH of i+3. The only strict requirement is a P in the i+1 position. A variety of SPKK motif-containing peptides have been shown to bind in the minor groove of DNA, with each turn binding 2 bp. Given the inherent limitations in comparing four residue motifs and the variety of SPKK motifs that can bind in the minor groove, the similarity of the ananassae oligonucleotide consensus to the SPKK motif is compelling (Malik, 2002).

Using the consensus created from the ananassae group, the MAST program was used to search the nonredundant database of proteins. Surprisingly, the top hit found was the mouse CenH3, the Cenp-A protein. Given that the search query was only nine amino acids long and that the match to Cenp-A was in the N-terminal tail, this match is especially noteworthy. In addition to mouse, matches to the ananassae group consensus were found in zebrafish and human Cenp-A N-terminal tails at approximately similar locations upstream of the histone fold domain. These matches were confirmed by a MAST search of a database consisting of 15 putative CenH3s obtained from various nucleotide databases. The ananassae consensus motifs were not found in CenH3s from fungi, nematodes, plants, or even D. melanogaster and D. pseudoobscura. Therefore, the ananassae-vertebrate sequence similarity likely represents an example of evolutionary convergence. In addition to the ananassae consensus repeats, the three Cenp-A sequences have an additional SPKK motif (GPRR in human and mouse Cenp-A) as well as recurring dipeptide motifs that include a proline at every other position (GP, SP, etc.) (Malik, 2002).

In S. cerevisiae, deletion analysis has shown that the N-terminal tail of CenH3 (Cse4) includes a 33-amino acid stretch (END domain) that is essential for function (Chen, 2000). By two-hybrid and dosage suppression of temperature-sensitive alleles, the END domain has been shown to interact with kinetochore components (Chen, 2000). Using an evolutionary approach, three conserved blocks have been identified in the N-terminal tail of Drosophila Cid. By analogy with END in Cse4, it is suggested that at least two of these three Cid blocks may also mediate interactions with other centromere-kinetochore determinants (Block 3 may interact with DNA). Unlike the single nucleosome centromere of S. cerevisiae, metazoan centromeres are hundreds of kilobases in length. Thus, some of the interactions involving Cid's N-terminal tail may occur between two successive Cid molecules in a centromeric nucleosome array. These blocks can now be used in protein interaction screens to elucidate the next level of centromere organization (Malik, 2002).

Adaptive evolution of Cid has been mapped to both the Loop 1 region of the histone fold domain as well as to multiple locations of the N-terminal tail in comparisons of D. melanogaster and D. simulans (Malik, 2001). It is proposed that altered DNA-binding specificity drives CenH3's adaptive evolution. Because this adaptation has occurred in multiple segments of Cid's N-terminal tail, the N-terminal tail must make extensive contacts with the linker DNA in centromeric chromatin (Malik, 2002).

One special instance of a likely CenH3-DNA interaction is evident in the case of SPKK-containing oligopeptides found in the ananassae species' Cid and vertebrate Cenp-A. Even in the absence of SPKK motifs, conventional histone tails interact extensively with the linker DNA. When found in H2A C-terminal or H2B N-terminal histone tails, SPKK-containing motifs confer an additional protection of 16 bp of linker DNA in reconstitution experiments. The N-terminal tails of H2B and H3 are perfectly positioned to interact with the minor groove of DNA as they exit the nucleosome in minor groove channels. Similarly, while the winged helix domain of linker histones is located at the nucleosome dyad, the positively charged N- and C-terminal tails interact extensively with the linker DNA. This interaction is weaker in rat testis H1 (H1t), which lacks SPKK motifs, compared with somatic H1s, which have them. Thus, the presence of minor groove interactions mediated by CenH3's N-terminal tail might result in uniform nuclease accessibility of centromeric chromatin. A comparison of H3 and H1 tails is particularly relevant as the H1 binding site on the nucleosome is very close to where the N-terminal tails of H3s exit. It is proposed that the minor groove binding repeats in CenH3 N-terminal tails play a role similar to that of H1 tails by neutralizing phosphates in the linker DNA -- i.e., they shield charges to allow superhelical bending and collapse into a higher order chromatin structure. This structure would be seen as the primary constriction, the cytological marker for centromere position. It will be interesting to determine whether centromeric chromatin is especially lacking in linker histones (Malik, 2002).

Two of the best characterized minor-groove binding motifs, SPKK and AT-hooks, show no sequence similarity to each other, suggesting that only a subset of potential minor-groove binding motifs has been explored. In addition to the SPKK-bearing motifs, additional types of repeats are found both in Cenp-A as well as in the different lineages of Drosophila, such as the takahashii consensus and NPKS expansions in D. orena, that may all carry out the same function -- i.e., bind in the minor groove of centromeric DNA. These peptides, like SPKK, are predicted to have a strong preference for AT-rich DNA, and may only recognize architectural DNA features such as minor groove shape rather than specific bases (Malik, 2002).

In conclusion, this examination of evolutionary constraint in the Drosophila CenH3 lineage has revealed that the N-terminal tail comprises a combination of interaction sites. Putative protein interaction blocks have been inferred that might lay the foundation for kinetochore formation, DNA binding regions subject to adaptive evolution, and nonspecific minor groove binding motifs. The resulting complex with DNA may form the universal cytological feature of centromeres, the primary constriction (Malik, 2002).

Cid homologs in yeast

A Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere (Stoler, 1995).

Direct evidence is presented that Cse4p, a histone H3 variant, is a structural component of the core centromere of S. cerevisiae. In histone H4 and Cse4p mutants, the core centromere chromatin structure is disrupted at restrictive temperature. Overexpression of Cse4p suppresses this defect in the H4 mutant, implying that the two proteins act together in centromere structure. Cse4p is specifically cross-linked to centromeric DNA. Furthermore, Cse4p is found in discrete foci consistent with that expected for centromeres. These results suggest the kinetochore is assembled on a specialized centromeric nucleosome containing Cse4p (Meluh, 1998).

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus show that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts results in cell lethality. However, all large lethal substitution alleles can be subdivided into smaller viable alleles, many of which cause elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. These findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A (Keith, 1999).

Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, measurements were made of the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, it was found that mutations throughout the Cse4p histone-fold domain cause surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but have no effect on chromosomes with CDE III mutant centromeres. This genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, a model is proposed that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome (Keith, 2000).

Cse4p is a structural component of the core centromere of Saccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. It has been proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, random mutations were targeted to the Cse4p histone fold domain and three temperature-sensitive cse4 alleles were isolated in an unbiased genetic screen. Two of the cse4 alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4 allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated (Glowczewski, 2000).

Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. A comprehensive and systematic mutagenesis of the Cse4p N terminus has been carried out to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation (Chen, 2000).

Mammalian kinetochores contain the centromere-specific histone H3 variant CENP-A, whose incorporation into limited chromosomal regions may be important for centromere function and chromosome segregation during mitosis. However, regulation of CENP-A localization and its role have not been clear. Reported here is evidence that the fission yeast homolog SpCENP-A is essential for establishing centromere chromatin associated with equal chromosome segregation. SpCENP-A binding to the nonrepetitious inner centromeres depended on Mis6, an essential centromere connector protein acting during G1-S phase of the cell cycle. Mis6 is likely required for recruiting SpCENP-A to form proper connection of sister centromeres (Takahashi, 2000).

Kinetochores are the specialized protein structures that form on centromeric DNA and direct chromosome segregation. It is critical that all chromosomes assemble a single kinetochore every cell cycle. One hallmark of all eukaryotic kinetochores is CENP-A, an essential centromeric histone H3 (CenH3) variant. Overexpression of CENP-A causes mislocalization to euchromatin, which could lead to deleterious consequences because CENP-A overexpression is associated with colorectal cancer. Although CENP-A protein levels are important for genomic stability, little is known about the mechanisms of CenH3 regulation. This study shows that the levels of the budding yeast CenH3, Cse4, are regulated by ubiquitin-proteasome-mediated proteolysis. Because mutation of all Cse4 lysine residues did not completely stabilize the protein, a dominant lethal mutant, CSE4-351, was isolated that was stable. The Cse4-351 protein localized to euchromatin, suggesting that proteolysis prevents CenH3 euchromatic localization. When wild-type Cse4 is fused to a degron signal (an N-terminal amino acid sequence that influences the degradation of a protein), the soluble Cse4 protein is rapidly degraded, but the centromere bound Cse4 is stable, indicating that centromere localization protects Cse4 from degradation. Taken together, these data identify proteolysis as one mechanism that contributes to the restricted centromere localization of the yeast CenH3 (Collins, 2004).

Cid homologs in invertebrates

The segregation of a chromosome during mitosis is mediated by a region of the chromosome known as the centromere, which organizes the kinetochore, to which the spindle microtubules attach. Many organisms have monocentric chromosomes, in which the centromeres map to single loci, whereas others, including the nematode C. elegans, have holocentric chromosomes, in which non-localized kinetochores extend along the length of each chromosome. The centromeres of monocentric chromosomes use specialized nucleosomes containing histone-H3-like proteins (known as CENP-A in mammals and Cse4 in the yeast Saccharomyces cerevisiae). A C. elegans histone-H3-like protein is necessary for the proper segregation of chromosomes during mitosis and identifies the centromeres of these holocentric chromosomes, indicating that both holocentric and monocentric chromosomes use centromeric histone-H3-like proteins (Buchwitz, 1999).

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, live and fixed assays were used in the one-cell stage Caenorhabditis elegans embryo. The consequences are compared of depleting homologs of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical 'kinetochore null' phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos has revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation (Oegema, 2001).

CENP-A homologs in vertebrates

The 17 kDa human autoantigen designated CENP-A is a centromere specific histone. CENP-A is present in tissue of bovine origin, and is quantitatively retained in mature spermatozoa. This result is striking, since a prominent feature of spermatogenesis in mammals is the replacement of most somatic and testes specific histones with protamines. Indirect immunofluorescence studies further show that CENP-A is retained in sperm nuclei in discrete foci, rather than being dispersed throughout the sperm head. These observations suggest that CENP-A is a functionally important component of centromeres, and that pre-existing CENP-A:DNA interactions are likely to be important in organizing the centromeres of the paternal genome during early embryogenesis (Palmer, 1990).

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly, but whose molecular role in kinetochore structure and function is unknown. The first time monospecific antisera has been raised to CENP-A, a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 and that resembles the yeast centromeric component CSE4. CENP-A is found to be concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A co-purifies with nucleosomes, these data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA. However, taken together, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. It is speculated that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres (Warburton, 1997).

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric 'state' on the DNA remains unclear. Replication timing of the prekinetochore domain of human chromosomes has been directly examined. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine it has been demonstrated that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrates that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation (Shelby, 2000).

Centromere protein A (Cenpa for mouse, CENP-A for other species) is a histone H3-like protein that is thought to be involved in the nucleosomal packaging of centromeric DNA. Using gene targeting, the mouse Cenpa gene has been disrupted and the gene has been demonstrated to be essential. Heterozygous mice are healthy and fertile whereas null mutants fail to survive beyond 6.5 days postconception. Affected embryos show severe mitotic problems, including micronuclei and macronuclei formation, nuclear bridging and blebbing, and chromatin fragmentation and hypercondensation. Immunofluorescence analysis of interphase cells at day 5.5 reveals complete Cenpa depletion, diffuse Cenpb foci, absence of discrete Cenpc signal on centromeres, and dispersion of Cenpb and Cenpc throughout the nucleus. These results suggest that Cenpa is essential for kinetochore targeting of Cenpc and plays an early role in organizing centromeric chromatin at interphase. The evidence is consistent with the proposal of a critical epigenetic function for CENP-A in marking a chromosomal region for centromere formation (Howman, 2000).

Centromere protein A (CENP-A) is a variant of histone H3 with more than 60% sequence identity at the C-terminal histone fold domain. CENP-A specifically locates to active centromeres of animal chromosomes and therefore is believed to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. CENP-A, highly purified from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitution system mediated by nucleosome assembly protein-1 (NAP-1). The structure of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H2B, and H4, and closed circular DNAs has the following properties. By atomic force microscopy, 'beads on a string' images were obtained that are similar to those obtained with nucleosomes reconstituted with four standard histones. DNA ladders with repeats of approximately 10 bp are produced by DNase I digestion, indicating that the DNA is wrapped round the protein complex. Mononucleosomes isolated by glycerol gradient sedimentation have a relative molecular mass of approximately 200 kDa and are composed of 120-150 bp of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B. Thus, it is concluded that CENP-A forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA (Yoda, 2000).

After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that near serine 10 of histone H3, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, peptide antibodies were generated against the putative phosphorylation site of CENP-A. ELISA, Western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression (Zeitlin, 2001).

Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. This study demonstrates that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments it has been shown that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation (Lomonte, 2001).

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome has been identified. Detailed sequence analysis indicates that this domain contains no centromeric alpha-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human alpha-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed (Lo, 2001a).

Centromere protein A (CENP-A) is an essential centromere-specific histone H3 homolog. Using combined chromatin immunoprecipitation and DNA array analysis, a 330 kb CENP-A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10) has been identified. This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through lower-resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP-A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP-A within centromere-specific nucleosomes. The DNA within this domain has a high AT-content comparable to that of alpha-satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid-S phase. Neocentromere formation is accompanied by a replication time lag around but not within the CENP-A binding region, with this lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres (Lo, 2001b).

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, CENP-A was experimentally mistargeted to non-centromeric regions of chromatin and whether other centromere-kinetochore components were recruited was determined. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, since the mistargeting of other components is not sufficient for assembly of the same complex. These results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres (Van Hooser, 2001).

CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. Centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C has been isolated from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies. The CENP-A/B/C complex is shown to be predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicates that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation has shown that MNase digestion releases a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes shows that most of the CENP-C and approximately half the CENP-B takes part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs shows that MNase digestion first releases the CENP-A/B/C chromatin complex into the soluble fraction, and later removes CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, it is proposed that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells (Ando, 2002).

The centromere is the chromosomal site that joins to microtubules during mitosis for proper segregation. Determining the location of a centromere-specific histone H3 called CENP-A at the centromere is vital for understanding centromere structure and function. Three human proteins have been identified essential for centromere/kinetochore structure and function, hMis18α, hMis18β, and M18BP1, the complex of which is accumulated specifically at the telophase-G1 centromere. Evidence is provided that such centromeric localization of hMis18 is essential for the subsequent recruitment of de novo-synthesized CENP-A. If any of the three is knocked down by RNAi, centromere recruitment of newly synthesized CENP-A is rapidly abolished, followed by defects such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei. Tricostatin A, an inhibitor to histone deacetylase, suppresses the loss of CENP-A recruitment to centromeres in hMis18α RNAi cells. Telophase centromere chromatin may be primed or licensed by the hMis18 complex and RbAp46/48 to recruit CENP-A through regulating the acetylation status in the centromere (Fujita, 2007).

Centromeres are chromosomal structures required for equal DNA segregation to daughter cells, comprising specialized nucleosomes containing centromere protein A (CENP-A) histone, which provide the basis for centromeric chromatin assembly. Discovery of centromere protein components is progressing, but knowledge related to their establishment and maintenance remains limited. Previously, using anti-CENP-A native chromatin immunoprecipitation, the interphase-centromere complex (ICEN) was isolated. Among ICEN components, subunits of the remodeling and spacing factor (RSF) complex, Rsf-1 and SNF2h proteins, were found. This paper describes the relationship of the RSF complex to centromere structure and function, demonstrating its requirement for maintenance of CENP-A at the centromeric core chromatin in HeLa cells. The RSF complex interacted with CENP-A chromatin in mid-G1. Rsf-1 depletion induced loss of centromeric CENP-A, and purified RSF complex reconstituted and spaced CENP-A nucleosomes in vitro. From these data, it is proposed the RSF complex as a new factor actively supporting the assembly of CENP-A chromatin (Perpelescu, 2009).

Dual recognition of CENP-A nucleosomes is required for centromere assembly

Centromeres contain specialized nucleosomes in which histone H3 is replaced by the histone variant centromere protein A (CENP-A). CENP-A nucleosomes are thought to act as an epigenetic mark that specifies centromere identity. CENP-N has been identified as a CENP-A nucleosome-specific binding protein. This study shows that CENP-C also binds directly and specifically to CENP-A nucleosomes. Nucleosome binding by CENP-C required the extreme C terminus of CENP-A and did not compete with CENP-N binding, which suggests that CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes. A mutation that disrupted CENP-C binding to CENP-A nucleosomes in vitro caused defects in CENP-C targeting to centromeres. Moreover, depletion of CENP-C with siRNA resulted in the mislocalization of all other nonhistone CENPs examined, including CENP-K, CENP-H, CENP-I, and CENP-T, and led to a partial reduction in centromeric CENP-A. It is propose that CENP-C binds directly to CENP-A chromatin and, together with CENP-N, provides the foundation upon which other centromere and kinetochore proteins are assembled (Carroll, 2010).

A model is proposed for human centromere assembly. A critical first step is the direct recognition of CENP-A chromatin by CENP-C. Because dimerization is important for CENP-C centromere targeting, but not for mononucleosome binding, it is speculated that each CENP-C dimer binds to two different, possibly adjacent, CENP-A nucleosomes within centromeric chromatin. A CENP-C dimer in conjunction with centromeric chromatin then provides the foundation upon which the rest of the Constitutive Centromere-Associated Network is assembled. CENP-N also binds directly to CENP-A nucleosomes, thus providing additional CENP-A nucleosome contacts that reinforce the specificity of the centromere assembly process (Carroll, 2010).

CENP-A phosphorylation by Aurora-A in prophase is required for enrichment of Aurora-B at inner centromeres and for kinetochore function

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylates CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference reveals that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. These observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function (Kunitoku, 2003).

The phosphorylation of CENP-A is involved in efficient occupancy of kinetochores with spindle fibers. Concurrent with CENP-A phosphorylation at early prophase, various proteins assemble at the outer domain of the kinetochore. Given that CENP-A is essential for this assembly process in several species, the phosphorylation of CENP-A on Ser-7 might be required to initiate it during prophase, before the kinetochores begin to attach to microtubules. Such protein recruitment triggered by CENP-A phosphorylation might be important for the establishment of kinetochore-microtubule connections. However, this modification does not appear to be necessary for generation of the spindle assembly checkpoint signal, because Mad2, BubR1, and CENP-E localizes normally to kinetochores in prometaphase cells expressing CENP-A(S7A) and these cells show a marked delay in prometaphase (Kunitoku, 2003).

Given that Aurora-B plays an important role in correcting kinetochore-microtubule attachment in mammalian cells, the mislocalization of Aurora-B might contribute to the defect in chromosome alignment in cells expressing CENP-A(S7A) or in those deficient in Aurora-A. However, because Aurora-A-mediated phosphorylation of CENP-A on Ser-7 during prophase appears to be important for microtubule attachment, it was not possible to assess the possible contribution of the attachment-correcting function of Aurora-B. The many misaligned chromosomes that were found in cells in which CENP-A phosphorylation was prevented possessed either unattached or monotelic kinetochores, whereas those in Aurora-B-depleted cells exhibited syntelic attachment. Further molecular dissection of the regulation of kinetochore function by Aurora kinases could be facilitated by identification of the proteins that are recruited to the kinetochore in a manner dependent on CENP-A phosphorylation on Ser-7 (Kunitoku, 2003).

Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP

In higher eukaryotes, the centromere is epigenetically specified by the histone H3 variant Centromere Protein-A (CENP-A). Deposition of CENP-A to the centromere requires histone chaperone HJURP (Holliday junction recognition protein). The crystal structure of an HJURP-CENP-A-histone H4 complex shows that HJURP binds a CENP-A-H4 heterodimer. The C-terminal β-sheet domain of HJURP caps the DNA-binding region of the histone heterodimer, preventing it from spontaneous association with DNA. This analysis also revealed a novel site in CENP-A that distinguishes it from histone H3 in its ability to bind HJURP. These findings provide key information for specific recognition of CENP-A and mechanistic insights into the process of centromeric chromatin assembly (Hu, 2011).

HJURP has been identified is distantly related to the yeast centromeric protein Scm3. HJURP interacts directly with CENP-A and histone H4, localizes CENP-A to the centromere in a cell cycle-dependent manner, and enables the deposition of newly synthesized CENP-A into the centromeric nucleosome. An approximately 80-amino-acid CENP-A-binding domain (CBD) at the N terminus of HJURP is necessary and sufficient for binding CENP-A, and a region encompassing loop-1 and helix α2 of the histone fold domain of CENP-A, known as the CENP-A targeting domain (CATD), is required for interaction with HJURP. This study provide the structural basis for the recognition of CENP-A by HJURP, as well as mechanistic insights into the histone chaperone activity of HJURP (Hu, 2011).

It is striking that the Ser 68 site outside of the CENP-A CATD plays a critical role for HJURP recognition, as previous studies have shown that the H3CATD chimera protein recapitulated essential functions of CENP-A, and the current in vitro results also confirmed direct binding of H3CATD to HJURP. Surprisingly, analyses of the HJURP-CENP-A-H4 and H3-H4 structures revealed no hindrance of HJURP binding in the histone H3 region corresponding to CATD. One scenario, which has been called a 'yin-yang' model, is that CATD provides major binding affinities for HJURP, and the Ser 68 site serves as a principal determinant of CENP-A specificity. In this model, the histone H3 region corresponding to the CATD of CENP-A can interact with HJURP, perhaps suboptimally, but Gln 68 pushes away HJURP; the latter force wins and there is no binding. In H3CATD, the artificially introduced CATD can overcome the energy barrier caused by the unfavorable contact of Gln 68, resulting in the binding of HJURP. Less intuitive in this mode is how the CATD overpowers Gln 68 in H3CATD, as the S68Q mutant of CENP-A with a native CATD loses the ability to bind HJURP. It should be pointed out that structural change may play an important role in the resolution of this puzzle, as it is evident from the deuterium exchange experiments that a non-CATD region in helix α1 of H3CATD has a different conformation from the corresponding region in histone H3 or CENP-A. It is also evident from structural comparisons that the α1-L1 region is the most variable region among the structures of histone H3-H4 and CENP-A-H4 complexes. Thus, it is possible that introduction of a CATD into H3 resulted in an environment that remedied the adverse effect of Gln 68. A cocrystal structure of HJURP CBD in complex with the H3CATD-H4 complex should clarify the role of Gln 68 in H3CATD, and, together with in vivo experiments, will provide further tests of the 'yin-yang' model of centromere targeting proposed in this study (Hu, 2011).

The discovery that HJURP binds a heterodimeric form of the CENP-A-H4 complex also has profound implications in understanding the molecular mechanism of assembly of CENP-A-containing nucleosomes. The precise model of CENP-A-containing nucleosomes is still a matter of debate. A closely relevant point here is whether a centromeric nucleosome contains a CENP-A-H4 heterodimer or heterotetramer. The structural result cannot distinguish whether a CENP-A nucleosome is octameric or a hemisome. However, it does point out that, if CENP-A nucleosomes were octameric, additional processes or regulations would be required to ensure that the CENP-A-containing nucleosomes are predominantly homotypic, as heterotypic nucleosomes with both CENP-A and histone H3 are a minority species (Hu, 2011).

In summary, this structural and biochemical analyses of the HJURP CBD-CENP-A-histone H4 complex have provided novel insights into the specificity of CENP-A recognition by HJURP, and advanced understanding of the histone chaperone activities of HJURP in preventing the formation of a CEN-A-histone H4 tetramer and modulating the DNA-binding activity of the CENP-A-histone H4 complex. The results of this study should facilitate in-depth analyses of the molecular mechanism of centromeric chromatin assembly (Hu, 2011).

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively (Fachinetti, 2013).

A long-standing question in chromosome inheritance is the epigenetic mark of centromere identity. By artificially targeting components to chromatin, several groups have demonstrated that large artificial arrays of CENP-A-containing chromatin are sufficient to generate partially functional centromeres, but only after completely abrogating any epigenetic component. An earlier effort had reduced CENP-A levels using siRNA. Such approaches fail to test the epigenetic question as they are plagued by partial suppression that precludes testing epigenetic sufficiency. Indeed, this study now demonstrates that as little as 1% of the original CENP-A level is sufficient for retention of at least partial centromere function and assembly of kinetochore proteins (Fachinetti, 2013).

Use of conditional gene inactivation in human cells and in fission yeast has overcome the previous technical limitations and has now established that the CATD of CENP-A when substituted into histone H3 can template its cell-cycle-dependent, HJURP/Scm3-dependent centromeric loading at a constant level in the complete absence of CENP-A. Consideration of the number of molecules of CENP-A at the normal centromere offers strong support for this conclusion. The number of CENP-A molecules at the 40–500 kilobase chicken centromeres has been reported to be between 25 and 40. Increasing that by tenfold to account for the increased centromere size in humans yields a maximal estimate of ~ 250–400 CENP-A molecules per centromere, with a corresponding prediction of <1 molecule remaining per centromere within 9 divisions after CENP-A gene inactivation. Therefore, the current evidence demonstrates that H3CATD continues to be loaded at its initial level at each centromere for 4–5 generations after the CENP-A level has fallen below ∼ 1 molecule per centromere. It is important to note that initial H3CATD assembly occurs in the presence of endogenous CENP-A; therefore, the evidence offers no insight into de novo centromere formation (Fachinetti, 2013).

Despite its necessity and sufficiency for maintaining centromere identity in the absence of CENP-A, this study has shown that H3CATD is not sufficient for long-term centromere function and cell viability because it does not nucleate kinetochore assembly. Rather, this study has identified two redundant pathways that function to initiate kinetochore assembly onto an epigenetically defined chromatin core containing the CATD. The evidence establishes that CENP-A-containing chromatin is the epigenetic mark that can identify, maintain and propagate human centromere function indefinitely through a conserved two-step mechanism in which it templates its own CATD-dependent replication and nucleates subsequent kinetochore assembly. Furthermore, it was demonstrated that the principles of epigenetic centromere inheritance and function are conserved from human to fission yeast (Fachinetti, 2013).

Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly

In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. This study used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. H4K20 monomethylation (H4K20me1; see Drosophila Histone H4) was found to be enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. It is concluded that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly (Hori, 2014).

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. This study performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. Replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, were shown to reduce the inheritance accuracy of CENP-A/Cnp1 (see Drosophila Centromere identifier) nucleosome positioning within centromere. Mechanistically, it was demonstrated that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, this study showed that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, this study showed that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome (Li, 2017).


centromere identifier : Biological Overview | Regulation | Developmental Biology | References

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